LAUSR

Clostridioides difficile infection presents an escalating clinical challenge due to the proliferation of hypervirulent and antibiotic‐resistant strains. The primary symptoms of disease, namely colitis and diarrhea, are induced by the release of two toxins: TcdA and TcdB. Targeting these toxins with peptide inhibitors provides an attractive therapeutic strategy that can be used alone or synergistically with standard antibiotic treatments to alleviate severe symptoms and reduce the risk of resistance development. In this study, we present the rational discovery and optimization of potent TcdB peptide inhibitors. The lead sequences effectively inhibit TcdB glucosyltransferase activity, the crucial enzymatic process leading to disease symptoms, by directly competing with the toxin's molecular targets, Rho proteins. Detailed enzymatic studies also elucidate distinct Michaelis constants, KM, for each substrate, UDP‐glucose and Rho‐proteins, for multiple TcdB GTD subtypes. The selected peptides demonstrated broad efficacy against the three most common TcdB subtypes, which are used in over 90% of clinical isolates. Additionally, the peptides delayed TcdB‐induced loss of barrier integrity and decreased apoptosis in a primary human colon epithelial monolayer model. This study highlights a novel therapeutic avenue with significant potential to enhance the treatment and management of C. difficile infections.